Lighting Up Microscopes: Advances and Emerging Sources

MARIE FREEBODY, CONTRIBUTING EDITOR, marie.freebody@photonics.com

Microscope developers are a resourceful bunch, opting to use the light source available to them at the time to peer at or below the surface of various materials. Even dating back to the 17th century, Galileo used sunlight to produce the very first optical microscope.

The next illumination sources to become available were lamps such as tungsten (incandescent), xenon, mercury and metal halide vapor. While such lamps can still be found in laboratories today, it was when an entirely new way of generating photons was discovered that the field of microscopy changed forever.

The year 1960 brought with it the invention of the laser. From then until today, researchers have been exploiting every variation of the laser and manipulations of its beam as they become available: from the gas laser to solid-state and the latest in quantum cascade laser (QCL)-based microscopy.

But it’s not just lasers in their various forms that dominate the field; an emergence of low-cost light-emitting diodes — originally made for general lighting — means that LEDs are increasingly being adopted.

Microscopy techniques in the material and life sciences can be broadly divided into three domains:

• Transmitted-light methods for transparent specimens that are typically in the form of a thin slice, for example, tens of microns thick.

• Reflected-light contrast methods for opaque specimens, such as metals, ceramics, plastics, minerals and wood, that remain opaque even when ground to a thickness of 30 microns.

• Fluorescence microscopy

LED lighting trend

For bright-field, transmitted-light techniques, a microscope is usually equipped with an incandescent tungsten-halogen or mercury arc lamp, which is effective and cheap. But in the last decade, LED light sources have increasingly displaced such lamps.

“Besides having a similar color rendering index [CRI], LEDs have a much longer lifetime — >25,000 hours versus approximately 2,000 hours — do not need any warm-up time, and show very little fluctuation during operation. LED light sources do also offer a higher degree of flexibility for integration into hardware and software,” said André Devaux, technical writer at PicoQuant GmbH, Berlin.

Leica DVM6 digital microscope images of embossed metal-coated paper used for food packaging. The images were acquired with different illumination contrast methods. LED full ring (a). Quarter of the ring light (b). LED coaxial light with polarizer open (c). LED coaxial light with polarizer closed (d). The quarter ring (b) and coaxial light with polarizer open (c) enhance the embossed squares, while the coaxial light with the polarizer closed (d) enhance the imperfections or contamination. Courtesy of Leica Microsystems.

Despite their development in the 1950s, LEDs have only recently become more popular in microscopy. As the lighting and automotive markets continue to drive the development of brighter, more efficient and cheaper LEDs, microscope manufacturers have begun to adopt LEDs into their products.

“Globally the halogen market has been declining in the past several years due to improving LED technology,” said Jennifer Wrigley, senior product manager of Industrial Microscopes at Olympus Scientific Solutions Americas. “The trend toward LED is a significant switch for the market from the traditional halogen.”

High-intensity LEDs provide similar intensities as halogen, but maintain a consistent color temperature regardless of intensity. This keeps the colors of features on the sample consistent for both viewing through the eyepieces and imaging with a digital camera.

Other advantages to the user are reduced cost of ownership — as LEDs use less energy than halogen; LEDs are more stable, durable and give off little heat, which is important when observing sensitive material; and LEDs can also be readily triggered to turn on and off in microseconds, without the need for shutters — a capability that has been tapped for fluorescence microscopy and live-cell imaging, and may find application in bright-field techniques as well.

Today’s LEDs can be manipulated to emit single color (narrow wavelength range) light from infrared to ultraviolet or can be combined in units to produce full spectrum white light.

Primary culture of rat cortical neurons, acquired with the Leica TCS SP8 X white light laser. Excitation and detection settings were adjusted to optimal image contrast. Courtesy of Leica Microsystems.

“White light illumination systems covering the spectral range from 350 to 700 nm are available from the major manufacturers and usually consist of 4 to 16 LEDs with different wavelengths,” said Devaux. “These systems also offer the advantage of fine-tunable intensity regulation, intensity stability over time and instant on/off switching.”

Advances and improvements over the next few years will most likely focus on better CRI, multiple color/wavelength emission, smaller size and so on.

“LEDs will probably become better in performance, depending on the microscopy application, cheaper and more practical to use. Some light colors/wavelengths emitted by LEDs are still relatively dim,” said Heinrich Bürgers, product manager for Stereo & Digital Microscopy at Leica Microsystems GmbH in Wetzlar, Germany. “For the case of fluorescence microscopy, a bright green LED is certainly needed.”

Stereo microscope image of a microelectronic circuit recorded with LED ring light illumination (a). Stereo microscope image of the same microelectronic circuit recorded with LED near-vertical illumination (b). Stereo microscope image of the same microelectronic circuit recorded with LED coaxial illumination (c). Courtesy of Leica Microsystems.

For widefield applications, Oliver Schlicker, application manager of Widefield Microscopy at Leica, has found that LEDs suitable for dyes in the far red range are more and more requested as red light penetrates much deeper into thicker samples with less stray light.

“Furthermore far red light has much less energy than shorter wavelengths, which is an advantage for long-term experiments with living samples,” he said.

Exploring laser light

Bright, nondivergent and available in a great variety of monochromatic wavelengths, it’s little wonder that lasers are the dominant light source for applications such as confocal and fluorescence microscopy. Two different laser types are used depending on the microscopy technique: continuous wave lasers for fluorescence microscopy and pulsed lasers for nonlinear or multiphoton microscopy, which require ultrashort pulses such as femtosecond lasers.

Confocal microscopy techniques based on laser excited fluorescence are key tools in the drive to connect molecular and cell biology. These images show simultaneous detection of endoplasmic reticulum localized soluble GFP and Golgi fusion protein Sec7-DsRed fusion proteins in the yeast S. cerevisiae. Image acquired with a Zeiss LSM 5 DUO at 20 frames/sec with simultaneous 488-nm and 561-nm excitation.Courtesy of Carissa Young, David Raden and Anne Robinson, University of Delaware.

Gas lasers were the first to be used, but in the mid-1990s they were mostly replaced by solid-state lasers starting with diode-pumped solid-state lasers. More recently, optically pumped semiconductor lasers and also laser diodes have begun to dominate, as diodes with useful visible output became available.

“Microscopy alone would probably not have spurred the completely new semiconductor materials needed for many of these laser diode wavelengths,” said Matthias Schulze, director of segment marketing for OEM Components and Instrumentation at Coherent Inc. in Santa Clara, Calif. “But consumer product market demand provided the necessary economic force.”

Diode-based lasers are especially valuable for any application requiring deep UV excitation, such as fluorescent lifetime imaging microscopy (FLIM), which exploits the natural emission of biomolecules. There are currently no LEDs available that could be used for this application.

Today, rapid progress in pulsed laser sources is giving access to more wavelengths (especially in the blue and deep UV), higher optical output powers, pulse widths and shapes optimized for specific applications and novel methods. Without these developments, super-resolution techniques such as stimulated emission depletion (STED), stochastic optical reconstruction microscopy, and photoactivated localization microscopy — which was recently honored with a Nobel Prize — would not have been possible.

“Pulsed laser sources continue to be in the focus [in] any time-resolved microscopy applications and the development of methods such as cross-correlation techniques,” said PicoQuant’s Devaux.

“Pulsed interleaved excitation methods and its combination with STED superresolution microscopy or its use in Förster resonance energy transfer studies progress rapidly,” he added. “These advances always call for laser sources with extended flexibility in wavelength, repetition rate, pulse width and generation of pulse sequences.”

An emerging trend in modern microscopy includes light sources with several laser lines. These “multilaser engines” provide laser light at several different wavelengths that can be controlled independently and simultaneously.

Toptica’s multilaser engine iChrome SLE provides eight laser lines out of one box. It has two output fibers with integrated switch, automated alignment and laser cartridges that enable exchanges in the field. Courtesy of Toptica.

“The integration of such a multilaser engine into a microscopy device is much easier than integrating one single laser for each line,” said Tim Paasch-Colberg, director of marketing at Toptica Photonics AG, Munich. “Also, the operation of the multilaser engine for the user is straightforward, since all different laser lines can be controlled with the same software interface.”

Tunable lasers are another area of development. For example, a supercontinuum laser — often referred to as white light laser — offered by Leica Microsystems or PicoQuant covers the spectrum from 470 to 670 nm. In combination with an acousto-optical beamsplitter, the Leica system offers up to eight different lines, which can be picked from the spectrum simultaneously and each line can be individually tuned in wavelength and intensity.

“Conventional laser sources provide only one or a small number of single wavelength lines. As a consequence, large gaps in the excitation spectrum limit the flexibility to excite nonstandard dyes and new fluorescent proteins,” said Jochen Sieber, product manager of Superresolution Technologies at Leica Microsystems.

Mid-infrared light source specialists Daylight Solutions, based in San Diego, has turned to another laser technology to provide a broadly tunable high-brightness source for next-generation benchtop infrared microscopes. Thanks to recent advances in quality, reliability and performance predictability, QCLs now provide brighter illumination for faster data acquisition in an entirely new type of microscope platform.

Current infrared microscopes are based on Fourier transform infrared (FTIR) technology, which use either the relatively weak light from a globar or synchrotron radiation from large particle accelerator facilities. Such sources require cameras that need cryogenic (liquid nitrogen)cooling to achieve adequate signal-to-noise ratio.

Daylight Solutions’ Spero microscope is the first and only commercially available QCL microscope on the market. Unlike in the FTIR method, not all wavelengths have to be scanned, which means the Spero provides rapid, high-resolution chemical imaging, which uses an uncooled microbolometer camera for a compact, benchtop instrument.

“The wide tunable range of the current generation QCLs is still limited, but by coupling several modules together it is possible to cover most of the infrared fingerprint spectral region, which contains most of the diagnostically useful information,” said Peter Gardner, professor of Analytical and Biomedical Spectroscopy at the Manchester Institute of Biotechnology, Manchester, U.K.

“Daylight Solutions [has] done this coupling four QCL modules to cover the approximate range 1900-900 cm⁻¹. They have then coupled this to an infrared microscope and a room temperature focal plane array detector.”

The last five years has seen huge investment in QCLs, which has helped boost quality, reliability and performance predictability of QCL-based systems.

“This investment was driven largely by the demanding requirements of our military-grade products, which are being tasked to protect aircraft from shoulder-fired missiles,” said Jeremy Rowlette, director of Molecular Imaging at Daylight Solutions. “The infrastructure built out to support these products helped raise the bar for all of our QCL products at Daylight.”

In truth, no single light source provides the optimum mix of performance and cost for every single microscopy application. Manufacturers must often offer multiple technologies and there is plenty of room for innovation in these light sources.

Today, both need and demand drive innovation. Researchers are multiplexing probes more, and mixing imaging modalities, even adding other analytical methods such as spectroscopy to their microscopy.

This means that light sources must keep up to enable these efforts by improving differentiation and signal-to-noise, and the demand should soon follow.

“We recognize the fundamental importance of optical microscopy. Although its roots go back hundreds of years, optical microscopy is growing faster than ever because of developments in life sciences: from well-funded initiatives like BRAIN in the area of neuroscience research, to pre-clinical and clinical diagnostic applications in support of modern higher life expectancy,” Coherent’s Schulze said. “We will continue to support these fields by providing the laser characteristics needed in these markets.”